Develop predictive human cardiomyocyte-based all optical assay for cardiotoxicity. Cardiotoxicity is the leading cause of safety-driven withdrawal a all stages and limitation of drug compounds. The current cardiac safety paradigm nonclinical guidance ICH S7B focuses on in vitro hERG assays, which prove to be suboptimal in predicting a compound's cardiotoxicity in human. In the context of high cost and low productivity for new drug discovery, better cardiac safety evaluation becomes a significant public health issue. CSRC, HESI, and FDA are developing a new cardio safety paradigm: Comprehensive In vitro Proarrhythmia Assay (CiPA), which comprises characterizing electrophysiological effects of compounds in iPSC-derived human cardiomyocytes. Q-State's platform can probe electrophysiology of such cells with higher throughput, lower cost, and higher information content (multi-modalities: voltage, Ca2+, pH, and ATP) than current patch clamp assays, and with better temporal and spatial resolution and lower phototoxicity than other optical screening tools. Supported by a $680K NINDS SBIR Phase I award and angel funding, Q-State Biosciences is developing a proprietary turnkey instrument and software system (Optopatch) for simultaneous optical perturbation and optical measurement of membrane voltage in neurons. We propose to adapt Optopach for cardiomyocyte measurements and to develop and validate an Optopatch assay for drug-induced cardiotoxicity in human iPSC- derived cardiomyocytes. Aim 1. Molecular tools. Select the most effective actuators (converting blue light into electrical stimuli for pacing) and reporters (converting AP waveforms into near infrared fluorescence signals) by comparing promoters, trafficking motifs and gene delivery methods, and to thoroughly characterize the sensitivity, speed, photostability, and repeatability of the molecular tools. Aim 2. Instrumentation. Adapt Optopatch hardware and software to optically stimulate and record from > 1,000 cardiomyocytes simultaneously. Develop fluidics and environmental controls; modify software to support optical pacing, image segmentation, and calculation of cardiac-relevant AP parameters. Aim 3. Assay development and testing. Optimize assays for acute and chronic cardiotoxicity. Validate the assay by quantifying AP changes under a panel of drugs with known mechanisms and cardiac safety profiles. We will provide non-GLP cardiotoxicity screening services to drug discovery companies at the lead identification and optimization stages, supplementing, with the potential to eventually replace, hERG assays. We will also make Optopatch-cardio instrumentation and reagents available to the academic community; to enable mechanistic studies and broaden the applications of electrophysiology platform.